Alterations in the protein and lipid secretory profile of steatotic hepatocytes promote insulin resistance in cultured muscle cells — ASN Events
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Alterations in the protein and lipid secretory profile of steatotic hepatocytes promote insulin resistance in cultured muscle cells (#144)

Ruth CR Meex 1 , Alex Morris 1 , Andrew Hoy 1 , Russell Brown 2 , Rob Goode 3 , Melissa Burk 2 , Peter Meikle 4 , Mark Malloy 5 , Matthew Watt 1
  1. Biology of Lipid Metabolism laboratory, Department of Physiology, Monash University, Clayton, Vic, Australia
  2. Department of Physiology, Monash University, Clayton, Vic, Australia
  3. Department of Biochemistry and Molecular Biology, Clayton, Vic, Australia
  4. Baker IDI Heart and Diabetes Institute , Melbourne, Victoria, Australia
  5. Department of Chemistry & Biomolecular Sciences, Australian Proteome Analysis Facility, Sydney, NSW, Australia

Background: Non-alcoholic fatty liver disease (NAFLD) is a common health problem that is associated with obesity. Liver steatosis is an early manifestation in the aetiology of NAFLD, which precedes the development of dyslipidemia and type 2 diabetes. We tested the hypothesis that protein and lipid signals originating from the steatotic hepatocyte may induce “cross-talk” with other tissues to modulate metabolic phenotypes.

Methods: Male C57Bl/6J mice were fed a low-fat (LFD) or high-fat diet (HFD) for 6-8 weeks to induce simple steatosis. Hepatocytes were isolated and cultured and the secreted products were collected in protein and lipid free media for analysis (i.e. conditioned media).

Results: The isolated HFD hepatocytes contained 9-fold more intracellular triacylglycerol than LFD hepatocytes. Quantitative analysis of the HFD hepatocyte lipid secretome detected 16 lipid classes and >400 lipid species, and showed increased secretion of 11 lipid classes (including cholesterol esters (+255%), ceramide (+149%), diglyceride (+247%), phosphatidylcholine (+207%), and triglyceride (+438%)). Secreted lipids correlated tightly with intracellular lipids (R2 = 0.89). iTRAQ analysis of the conditioned media detected 538 proteins of which 115 were identified as ‘classically secreted’. Thirty-four proteins were differentially secreted in HFD vs. LFD hepatocytes. Transcriptomic analysis of the hepatocytes detected 11,526 genes of which 532 were identified as ‘classically secreted’. The overlap of the predicted and actual secretome was poor. Enrichment analysis of the protein data revealed that differently secreted proteins in the secretome of HFD vs. LFD were involved in inflammatory processes, lipid metabolism, the metabolic syndrome, and insulin resistance. Conditioned media from HFD hepatocytes increased fatty acid uptake, oxidation and storage, and caused insulin resistance in L6Glut4-myotubes. There were negligible effects in adipocytes and macrophages.

Conclusion: Hepatic steatosis invoked changes in the protein and lipid secretory profile that, in turn, altered the metabolic phenotype of muscle and decreased insulin sensitivity. 

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